Cannabichromene is a cannabinoid CB2 receptor agonist

Article abstract of DOI:10.1111/bph.14815

Background and Purpose: Cannabichromene (CBC) is one of the most abundant phytocannabinoids in Cannabis spp. It has modest antinociceptive and anti-inflammatory effects and potentiates some effects of Δ⁹-tetrahydrocannabinol in vivo. How CBC exerts these effects is poorly defined and there is little information about its efficacy at cannabinoid receptors. We sought to determine the functional activity of CBC at cannabinoid CB₁ and CB₂ receptors. Experimental Approach: AtT20 cells stably expressing haemagglutinin-tagged human CB₁ and CB₂ receptors were used. Assays of cellular membrane potential and loss of cell surface receptors were performed. Key Results: CBC activated CB₂ but not CB₁ receptors to produce hyperpolarization of AtT20 cells. This activation was inhibited by a CB₂ receptor antagonist AM630, and sensitive to Pertussis toxin. Application of CBC reduced activation of CB₂, but not CB₁, receptors by subsequent co-application of CP55,940, an efficacious CB₁ and CB₂ receptor agonist. Continuous CBC application induced loss of cell surface CB₂ receptors and desensitization of the CB₂ receptor-induced hyperpolarization. Conclusions and Implications: CBC is a selective CB₂ receptor agonist displaying higher efficacy than tetrahydrocannabinol in hyperpolarizing AtT20 cells. CBC can also recruit CB₂ receptor regulatory mechanisms. CBC may contribute to the potential therapeutic effectiveness of some cannabis preparations, potentially through CB₂ receptor-mediated modulation of inflammation.

Full text of DOI:10.1111/bph.14815

Connor Mark (Orcid ID: 0000-0003-2538-2001)
Cannabichromene is a cannabinoid CB2 receptor
agonist
Michael Udoh, Marina Santiago, Steven Devenisha, Iain S. McGregora, and
Mark Connor.
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences,
Macquarie University, aLambert Initiative for Cannabinoid Therapeutics, Brain and Mind
Centre, The University of Sydney; Sydney, Australia.
Correspondence: Mark Connor, Department of Biomedical Sciences, Macquarie
University, Sydney, Australia. E-mail: mark.connor@mq.edu.au
Running Title: Cannabichromene is a CB2 receptor agonist.
What is already known:
 The phytocannabinoid cannabichromene (CBC) has anti-nociceptive and anti-
inflammatory effects in vitro and in vivo
 How CBC exerts these effects is largely unknown
What this study adds:
 This study shows that CBC is a selective CB2 receptor agonist
 CBC has a higher in vitro efficacy than tetrahydrocannabinol, and activates CB2
receptor regulatory pathways
Clinical Significance:

Cannabis contains a CB2-selective compound that could reduce inflammation without
producing intoxication
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/bph.14815
This article is protected by copyright. All rights reserved.

BACKGROUND
Cannabichromene (CBC) is one of the most abundant phytocannabinoids in Cannabis
spp. It has modest anti-nociceptive and anti-inflammatory effects and potentiates some
effects of 9- tetrahydrocannabinol (THC) in vivo. How CBC exerts these effects is poorly
defined and there is little information about its efficacy at cannabinoid receptors. We
sought to determine the functional activity of CBC at CB1 and CB2 receptors.
EXPERIMENTAL APPROACH
AtT20 cells stably expressing HA-tagged human CB1 and CB2 receptors were used.
Assays of cellular membrane potential and loss of cell surface receptors were performed.
KEY RESULTS
CBC activated CB2 but not CB1 receptors to produce a hyperpolarization of AtT20 cells.
This activation was inhibited by a CB2 antagonist AM630, and sensitive to pertussis toxin.
Application of CBC reduced activation of CB2 receptors (but not CB1 receptors) by
subsequent co-application of CP55,940, an efficacious CB1 and CB2 agonist. Continuous
CBC application induced loss of cell surface CB2 receptors and desensitisation of the
CB2-induced hyperpolarization.
CONCLUSIONS AND IMPLICATIONS
CBC is a selective CB2 receptor agonist displaying higher efficacy than THC in
hyperpolarising AtT20 cells. CBC can also recruit CB2 receptor regulatory mechanisms.
CBC may contribute to the potential therapeutic effectiveness of some cannabis
preparations, potentially through CB2-mediated modulation of inflammation.
Keywords Cannabichromene, phytocannabinoid, cannabinoids, desensitisation, surface
receptor internalisation.
This article is protected by copyright. All rights reserved.

Abbreviations
2-AG, 2-arachidonoyl-glycerol; AEA, anandamide; AtT20-CB1, Mouse pituitary tumour
cells stably transfected with HA human CB1 cells; AtT20-CB2, Mouse pituitary tumour
cells stably transfected with HA human CB2 cells; CB1, Cannabinoid receptor type 1,
CB2, Cannabinoid receptor type 2; CBC, cannabichromene; CBD, Cannabidiol; CBN,
Cannabinol; ECS, Endocannabinoid system; GRK, G-protein coupled receptor kinase;
HA, Haemagglutinin, PTX, Pertussis toxin; THC, Tetrahydrocannabinol; THCV,
Tetrahydrocannabivarin.
This article is protected by copyright. All rights reserved.

Introduction
Cannabichromene (CBC) is one of over 100 phytochemicals, (collectively referred to as
phytocannabinoids) that are found in Cannabis spp (ElSohly  Gul, 2014). CBC was
identified in 1966 and is one of the most abundant phytocannabinoids alongside 9-
tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) (Izzo et al, 2009;
Turner, Elshohly & Boeren, 1980). Evaluation of seized cannabis plants in USA, UK and
Australia showed CBC concentrations ranging between 0.05 - 0.3%w/w (Mehmedic et
al., 2010; Potter, Clark, & Brown, 2008; Swift et al., 2013). CBC, THC and CBD are
directly synthesized from cannabigerolic acid, and share a common 3-pentylphenol ring
(Fig.1) (Flores-Sanchez & Verpoorte, 2008). The therapeutic potential of CBC has been
evident in several preclinical studies: for example, CBC decreased carrageenan-induced
and lipopolysaccharide (LPS)- induced inflammation in rats and mice, respectively
(Turner & Elsohly 1981; DeLong et al. 2010); modestly inhibited thermal nociception
and potentiated THC anti-nociception in mice (Davis & Hatoum 1983; DeLong et al.
2010). While this may be mediated in part through changes in THC distribution in the
mice (DeLong et al. 2010), the pharmacological basis for the in vivo actions of CBC
remains unclear.
The endocannabinoid system (ECS) comprises the cannabinoid receptors (CB1 and
CB2), endogenous agonists (anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG)),
putative endocannabinoid transporters, enzymes involved in the synthesis and
metabolism of endocannabinoids (Iannotti et al, 2016). Cannabinoid receptors are
differentially distributed in the body. CB1, the most abundant GPCR in the mammalian
brain (Marsicano & Lutz, 1999), is predominantly expressed in the central nervous
system; while CB2 is expressed abundantly in cells of the immune system and organs
This article is protected by copyright. All rights reserved.

such as the spleen. These distributions imply that activation of these receptors will induce
different physiological responses. For example, THC causes a distinctive intoxication via
stimulation of the CB1 receptors, while stimulation of CB2 receptors does not appear to
contribute to the psychoactive effects of cannabis (Deng et al., 2015).
Phytocannabinoids target individual components of the ECS and act on a range of other
receptors and ion channels. For example, THC activates CB1 and CB2 receptors, but also
modulates GPR55, 5HT3A receptors and peroxisome proliferator-activated receptor
gamma (PPARγ) (Bayewitch et al. 1996; Pertwee 1999; Barann et al. 2002; O’Sullivan
et al. 2005; Ryberg et al. 2007; Lauckner et al. 2008). CBD is reported to increase
anandamide levels by inhibiting the enzyme FAAH; act as a negative allosteric modulator
of CB1 receptors;antagonise GPR55 receptors; activate TRPV2 receptors, and modulate
T-type calcium channels(Ross et al., 2008; De Petrocellis et al., 2011; Laprairie et al.,
2015). The less prevalent phytocannabinoids such as tetrahydrocannabivarin (THCV), is
a low efficacy CB2 agonist, and high potency TRPV1 and TRPA1 agonist (De Petrocellis
et al., 2011), while cannabinol (CBN) appears to be an agonist of CB1, CB2 receptors
and TRPA1 channels (Bolognini et al., 2010; De Petrocellis et al., 2011; Rhee et al.,
1997).
CBC has been reported to be a low affinity CB1/CB2 ligand in binding assays conducted
on human receptors expressed in insect cells (Rosenthaler et al., 2014), and it also
activates rat TRPA1 channels (De Petrocellis et al., 2011). However, receptor binding
does not provide information about ligand efficacy, andwhetherCBC has efficacyat either
receptor remains unresolved. In this study, we sought to characterise the action of CBC at
human CB1 and CB2 receptors. To do this, we used an in vitro assay of inwardly
rectifying potassium channel activation in intact AtT-20 cells, that we have used
extensively to characterize the activity of cannabinoids at CB1 and CB2 receptors
This article is protected by copyright. All rights reserved.

(Banister et al. 2016; Redmond et al. 2016; Soethoudt et al. 2017; Longworth et al. 2017).
Using this assay, we find that CBC is an agonist at CB2 but not CB1 receptors.
Materials and Methods
Compounds
CBC was synthesized according to the method of Lee & Wang (2005). Olivetol (1.80 g, 10
mmol) and citral (1.83 g, 12 mmol) were dissolved with stirring in toluene (100 mL), followed
by the addition of ethylenediamine (267 uL, 240 mg, 4 mmol) and acetic acid (458 uL, 480
mg, 8 mmol). The mixture was refluxed for 6 h and concentrated under vacuum. The residue
was dissolved in dichloromethane (DCM), washed with water and brine, filtered through a plug
of silica, and concentrated. Column chromatography was performed multiple times, as separate
runs utilising hexane with DCM (gradient from 5:1 to 1:1) and hexane with ethyl acetate or
acetone (preferably acetone; gradient from 67:1 to 50:1) were necessary to remove impurities.
Cannabichromene was afforded as a pale orange oil (1.20 g, 3.8 mmol, 38%), which darkened
upon exposure to air and light. 1HNMR (CDCl3, 400 MHz) δ 6.66 (1H, d, J = 9.9 Hz), 6.29
(1H, s), 6.14 (1H, s), 5.51 (1H, d, J = 9.9 Hz), 5.37 (1H, s), 5.12 (1H, t, J = 6.9 Hz), 2.44 (2H,
t, J = 7.67), 2.22-2.07 (2H, m), 1.81-1.66 (2H, m), 1.69 (3H, s), 1.60 (3H, s), 1.59-1.52 (2H,
m), 1.41 (3H, s), 1.37-1.25 (4H, m), 0.90 (3H, t, J = 6.6 Hz); 13CNMR (CDCl3, 100 MHz) δ
153.9, 151.1, 144.8, 131.6, 127.2, 124.2, 116.9, 109.1, 107.9, 107.1, 78.3, 41.0, 35.9, 31.5,
30.6, 26.2, 25.7, 22.7, 22.5, 17.6, 14.0. structure and purity of cannabichromene (>95%) was
confirmed using 1H and 13C NMR and LCMS. All physical and spectral properties were
consistent with those previously reported (Lee & Wang, 2005).
THC was obtained from THCPharm (Frankfurt, Germany) while CP55,940, SR141716
and AM630 were purchased from Cayman Chemical (Michigan, USA). All drugs were
This article is protected by copyright. All rights reserved.

prepared as stock solutions in DMSO and diluted using a 0.01% bovine serum albumin
(BSA, Sigma, Castle Hill, Australia) in HEPES-buffered low potassium Hanks Balanced
Salt Solution (HBSS). HBSS comprises (mM) NaCl 145, HEPES 22, Na2HPO4 0.338,
NaHCO3 4.17, KH2PO4 0.441, MgSO4 0.407, MgCl2 0.493, Glucose 5.56, CaCl2 1.26;
(pH7.4, Osmolarity 315±15mosmol). Final DMSO concentration was 0.1%.
Cell Culture
Mouse pituitary tumour AtT20 FlpIn cells stably transfected with HA-tagged human CB1
(AtT20-CB1) and human CB2 (AtT20-CB2) receptors (Alexander et al. 2017; Banister,
et al. 2016) were used. AtT20 FlpIn cells were made in our lab from AtT20 cells obtained
from the American Type Culture Collection (ATCC, RRID:CVCL_4109), using the Flp
In System from Thermo Fisher Scientific (#K601001). Tissue culture media and reagents
were from Thermo Fisher Scientific, (Massachusetts, USA) or Sigma-Aldrich (Castle
Hill, Australia). Tissue culture wares were sourced from Corning (Corning, NY, USA) or
Becton Dickinson (North Ryde, Australia). Cells were cultured in T75 flasks using
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine
serum (FBS) and 1% penicillin-streptomycin (100U/ml) and incubated in a humidified
atmosphere with 5% CO2 at 37˚C. Zeocin (100 µg/ml, Invivogen, California, USA), and
hygromycin (80u µg/ml, Invivogen) were used to select wild-type and transfected AtT20
cells, respectively. Cells were passaged at 80% confluency and used for assays at above
90% confluency, for up to 15 passages. For experiments, AtT20 cells were resuspended
in Leibovitz’s L-15 media containing 1% FBS, 1% P/S and 15mM glucose. 90 µL of the
resuspended cells were plated in a black-walled, 96-well microplate (Corning, NY, USA)
and incubated overnight in humidified air at 37˚C. For experiments involving pertussis
toxin treatment (PTX, Hello Bio, Bristol, UK), 200 ng/ml PTX was added to the L-15 cell
This article is protected by copyright. All rights reserved.

suspension
Membrane Potential Assay
In this assay, a reduction in fluorescence is indicative of cellular hyperpolarisation.
Changes in the membrane potential of cells were measured in duplicate, using a FLIPR
Membrane Potential Assay kit (blue #R8034), and a FlexStation 3 Microplate Reader
(both from Molecular Devices, Sunnyvale, CA). The dye was diluted to 50% of the
manufacturers recommended concentration using HBSS. Dye (90 µL) was loaded into
each well of the plate and incubated for 1 h at 37˚C prior to testing. The FlexStation 3
recorded fluorescence at 2 sec intervals (ʎexcitation= 530, ʎemission= 565), and drugs were
added after an initial 2 mins of baseline reading. The volume of each drug addition was
20 µL, and when two drug additions were made, each drug concentration was adjusted to
accommodate the change in final volume. The cellular response to the drug is presented
as a percentage change in fluorescence from baseline after subtraction of the change
produced by vehicle addition. The change in fluorescence was then normalized to the
change in fluorescence due to 1 µM CP55,940 (a high efficacy, non- selective CB1 and
CB2 receptor agonist) (Banister et al. 2016) to allow more ready comparison across
experiments. CP55,940 (1 µM) standard stimuli were obtained in independent
experiments in one well of each column of each plate. Concentration-response curves
were fitted to a 4-parameter sigmoidal dose-response curve in Graph Pad Prism (Version
6 GraphPad Software Inc, CA, USA; RRID:SCR_002798) to derive pEC50 and Emax
.
Receptor Internalisation Assay
Changes in cell surface CB2 receptors were determined in at least 5 independent
experiments, each performed in triplicate, using whole cell enzyme-linked immunosorbent
assay (ELISA). Cells in L-15 media were seeded at 80,000 cells per well in a Poly-D-
This article is protected by copyright. All rights reserved.

lysine (Sigma, Castle Hill, Australia) coated, black walled, clear bottom 96-well plate,
and incubated for 18 h at 37˚C in humidified air. After incubation, cells were treated with
the drug of interest. Reported drug concentrations are final concentrations. For one drug
treatment, the volume of cells in L-15 and compounds were mixed in a 1:1 ratio. For two
drug treatments, the volume of cells in L-15, drug A and drug B were added in ratio 9:9:2.
Following drug treatment, receptor trafficking was inhibited by placing cells on ice. Cells
were then fixed with 4% paraformaldehyde (PFA) for 15 mins. Fixed cells were washed
three times with 100 µL Phosphate Buffered Solution (PBS) and blocked with 1% BSA
in PBS for 1 h at room temperature. Alexa Fluor® 488 anti-HA Epitope Tag Antibody
(Biolegend, UK; RRID: AB_2565072), diluted to 1:250 with blocking solution, was
incubated with the cells at 4˚C for 18 h. Cells were then washed three times with 100 µL
PBS followed by the addition of 50 µL PBS for the quantification of fluorescence
intensity using PHERAstar plate reader (BMG Labtech, Germany). Loss of cell surface
receptor was calculated as the percentage decrease in fluorescence intensity after the
subtraction of background fluorescence (the fluorescence of wild-type AtT20 cells
incubated with the Anti-HA antibody, as above). The background fluorescence was 50 ±
3% of total fluorescence in CB2 expressing cells.
Data Analysis
Data analysis for the immunohistochemistry was blinded. For the membrane potential
assay experiments, blinding is impractical, but very effort was made to vary the location
within the plate and the order in which drugs were added to 96 well plates to minimize
the potential confounds of evaporation or unequal exposure time to the MPA dye. All
statistical analyses were conducted with GraphPad Prism, in line with the
recommendations on experimental design and analysis in pharmacology (Curtis et al.,
2015). Data are reported as Mean ± SEM. The equation used to fit a 4-parameter
This article is protected by copyright. All rights reserved.

sigmoidal dose-response is Y= Bottom
+
(Top-Bottom)/(1+10^((LogEC50-
X)*HillSlope)). Two-tailed, unpaired t-tests were used to compare two data points and
one-way ANOVA for more than two data points with Tukey post-hoc analysis. P-values
< 0.05 were considered statistically significant and indicated with * graphically. Unless
otherwise stated, five independent replicates were performed for each experiment.
Nomenclature of Targets and Ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in
http://www.guidetopharmacology.org the common portal of data from the IUPHAR/BPS
Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in
the Concise Guide to PHARMACOLOGY 2017/2018 (Alexander et al., 2017).
Results
CBC evokes cellular hyperpolarisation via CB2 but not CB1 receptors
CP55,940, a non-selective CB1/CB2 receptor agonist, produced a concentration-
dependent decrease in fluorescence in CB1 cells, with a pEC50 of 7.8 ± 0.1 (Fig. 2A).
THC also evoked membrane hyperpolarisation but with a lower efficacy and potency
(pEC50 of 6.6 ± 0.2; Emax of 53 ± 3% of CP55,940) (Fig 2A). CBC did not hyperpolarize
CB1 cells, inducing a negligible change in fluorescence of 2 ± 1% at 30 µM (Fig. 2A-B).
In CB2 cells, CP55,940 produced a concentration-dependent decrease in fluorescence
(pEC50 of 7.1 ± 0.1) (Fig. 2C). The maximum effect of THC (10 µM) was 22 ± 3% of
CP55,940 (Fig. 2C). In contrast to AtT20-CB1 cells, application of CBC to AtT20-CB2 cells
resulted in a significant hyperpolarisation, reaching a maximum of 52 ± 4% of the maximum
effect of CP55,940 at the highest concentrationof CBC tested (30 µM, Fig. 2C-D). 30 µM
CBC produced a negligible change in fluorescence when applied to wild-type AtT20 cells (2
This article is protected by copyright. All rights reserved.

± 0.4%).
CBC-induced hyperpolarisation is blocked by AM630 and is pertussis sensitive
Pre-treatment of AtT20-CB2 cells with AM630 (3 µM, 5 mins), a CB2 receptor selective
antagonist (Ignatowska-Jankowska, Jankowski, & Swiergiel, 2011), inhibited the 10 µM
CBC response by 93 ± 6% compared to vehicle pre-treated cells (Fig. 3A-B). AM630
similarly inhibited the responses to CP55,940 (300 nM, Fig. 3B). Overnight incubation of
CB2 cells with 200 ng/ml pertussis toxin strongly reduced responses to 10 µM CBC (8 ±
4% of CP 55,940) and 1 µM CP55,940 (11 ± 4%).
CBC inhibits CP55,940 responses at CB2 receptors, but not CB1 receptors
The effect of CBC on responses to CP55,940 and THC was investigated by pre-incubating
cells with CBC (10 µM, 5 mins). In CB1 cells, CBC did not significantly affect the
subsequent response to either CP55,940 (100 nM) or THC (10 µM) (Fig. 4A-B). In CB2
cells, theCP55,940 (300 nM) response was significantly inhibited by prior application of
CBC (10 µM, 5 mins, 44 ± 5%) (Fig. 4C & 4D) or CP55,940 (100 nM, 5 mins, 23 ± 5%)
(Suppl Fig 1A, B) respectively. Simultaneous application of CP55,940 (300 nM) and
CBC (10 µM) produced a similar change fluorescence to application of CP55,940 (300
nM) alone (112 ± 5 %, Suppl Fig 1C, D).
CBC treatment causes cell surface loss of CB2 receptors
CB2 receptors undergo agonist-induced loss of surface receptors following prolonged
stimulation (Grimsey et al, 2011). We found that 1 µM CP55,940 internalized CB2
surface receptors to 59 ± 3% of basal surface level (BSL) after 30 mins treatment. CBC
also internalized CB2 surface receptors (10 µM, 77 ± 5%; 30 µM, 71 ± 3%). When CB2
This article is protected by copyright. All rights reserved.

cells were pre-treated with AM630 (3 µM, 5 mins), 10 µM CBC did not produce
significant loss of surface CB2 receptors after 30 mins treatment (105 ± 4% of BSL) (Fig.
5A). The amount of cell surface receptors did not change when the cells were exposed to
AM630 (3 µM), followed by vehicle for 30 minutes (97 ± 8% of BSL). To assess the
possible role of G protein receptor kinases in CBC-mediated receptor internalisation, we
pre-treated cells with Compound 101 (10 µM), a GRK2/3 kinase inhibitor (Lowe et al.,
2015), for 1 h and then challenged them with CBC. There was no significant change in 10
µM CBC internalisation of CB2 surface receptors following Compound 101 pre-
treatment (Fig. 5B).
Effect of CBC on CB2 signalling desensitisation
In the membrane potential assay, prolonged stimulation of AtT20 cells expressing
cannabinoid receptors results in the slow reversal of cellular hyperpolarisation (Cawston
et al., 2013). Continuous stimulation of CB2 receptors for 30 mins by 1 µM CP55,940 or
10 µM CBC resulted in a reversal of the cellular hyperpolarization by 88 ± 3% (Fig 6A)
and 73 ± 6%, (Fig 6B-C), respectively. The desensitisation did not change significantly
when cells were pre-treated with Compound 101 (10 µM, 60mins) (Fig 6A-C).
Discussion
In this study, we have discovered that CBC is a phytocannabinoid with selective CB2
receptor agonist actions. We have also provided evidence that it signals through the Gi/o
type G-proteins, induces CB2 receptor internalisation and signalling desensitisation that
is independent of GRK2/3 kinases.
CBC produced a dose-dependent cell activation indicated by cellular hyperpolarisation
in CB2 cells but with no analogous hyperpolarisation in CB1 cells. This is consistent with
This article is protected by copyright. All rights reserved.

a previous finding that CBC apparently does not stimulate [35S]GTPγS binding via CB1
expressed in CHO cells (Romano et al., 2013) or inhibit AC activity in N18 cells natively
expressing mouse CB1 receptors (Howlett, 1986). Although no cannabinoid-antagonist
dependent effects have been elucidated in other assays, CBC has been reported to weakly
inhibit cellular AEA uptake and the 2-AG hydrolysing enzyme monoacylglycerol lipase
(MAGL), both of which may conceivably lead to an indirect activation of cannabinoid
receptors through increase in extracellular endocannabinoids (Ligresti, 2006; De
Petrocellis et al., 2011). However, the rapid onset of cellular hyperpolarisation in CB2
cells upon addition of CBC suggests a direct receptor activation. Our findings are also
consistent with previous studies which concluded that CBC does not significantly affect
the CB1 receptor mediated psychoactive effects of cannabis in-vivo (DeLong et al., 2010;
Ilan et al., 2005).
Cannabinoid receptors mediate downstream signalling predominantly through the Gi/o
protein family (Mallipeddi, et al, 2017), but CB1 can couple Gs-proteins when there is no
functional Gi/o-coupling (Bonhaus et al., 1998; Glass & Felder, 1997), and affect Gq in
some environments (Lauckner, Hille, & Mackie, 2005). The loss of CBC signalling upon
PTX treatment confirms Gi/o-protein coupling in the hyperpolarization assay, consistent
with previous findings with these cells (Banister et al., 2016).
CBC-induced hyperpolarisation in CB2 cells was absent in wild type AtT20 cells and
blocked by the selective CB2 receptor antagonist AM630. This blockade is likely due to
competitive binding at the CB2 receptor site, supporting the hypothesis that CBC effects
are mediated through the CB2 receptor orthosteric site. It is noteworthy that SR144,528,
a CB2 antagonist, does not block the anti- inflammatory effects of CBC either in vitro
(inhibition of nitrite formation in peritoneal macrophages) or in vivo (LPS-induced paw
oedema) assays (DeLong et al., 2010; Romano et al., 2013). The receptor mechanisms
This article is protected by copyright. All rights reserved.

underlying these anti-inflammatory effects are not yet fully defined.
THC is a low efficacy agonist in many assays of CB1 and CB2 receptor function
(Bayewitch et al., 1996; Soethoudt et al., 2017). Therefore, we investigated whether CBC
could be acting as an antagonist at the CB1 receptor, since it had been previously reported
to bind at the CB1 receptors, albeit with lower affinity than CB2 receptors (Rosenthaler
et al., 2014). Using sub-maximal concentrations of a high efficacy agonist (300 nM
CP55,940), and maximum concentration of a lower efficacy agonist (10 M THC), we
found that CBC did not alter the onset, and extent, of cellular hyperpolarisation in CB1
receptor expressing cells. These suggest that CBC does not significantly interact with the
CB1 receptor site. However, CBC significantly reduced the extent of CP55,940-induced
hyperpolarization in CB2 cells after 5 mins treatment. This is likely due to receptor
desensitisation, as CBC (10 µM) and CP55940 (300 nM) added at the same time
produced a similar effect to CP55940 (300 nM) alone. We showed that lower
concentration of CP55940 (100 nM) also produced a modest degree of desensitisation to
subsequent addition of a higher concentration (300 nM) of same drug.
Stimulation of both CB1 and CB2 receptors have been implicated in antinociception
(Bisogno et al., 2009; Guindon, Desroches, & Beaulieu, 2007; Kinsey et al., 2010; La
Rana et al., 2006; Lichtman et al., 2004). CB1 receptors are involved in the attenuation
of synaptic transmission of nociception in the brain and primary afferent neurons, while
CB2 contributes to antinociception by inhibiting the release of proinflammatory factors
around nociceptive neuron terminals (Manzanares, Julian, & Carrascosa, 2006). Since
THC analgesia is at least partly mediated through CB1 receptors (Mao et al., 2000) and
CBC is a ligand for CB2 receptors, it is possible that the potentiation of THC analgesia
by CBC, in addition to pharmacokinetic interaction (Davis & Hatoum, 1983; DeLong et
al., 2010) may be a result of CBC stimulation of CB2-mediated inhibition of the release
This article is protected by copyright. All rights reserved.

of pro- inflammatory factors. Apart from CB2-related anti-inflammatory activities, CBC
may act directlyor indirectly on proteins such as TRPA1 or adenosine A1 receptors (De
Petrocellis et al., 2008; Maione et al., 2011; Shinjyo & Di Marzo, 2013).
Upon sustained exposure to agonists, CB2 receptors undergo receptor internalisation,
resulting in signalling desensitisation (Bouaboula, Dussossoy, & Casellas, 1999;
Shoemaker et al, 2005). Our results show that CBC caused both loss of surface receptors
and signalling desensitisation of CB2 receptors. However, the loss of cell surface
receptors was less than that observed with CP55,940. Thismay be due to lower efficacy
of CBC in comparison to CP55,940, which is among the most efficacious cannabinoids
for internalisation (Atwood et al., 2012). CBC-induced loss of surface CB2 receptors was
antagonised by AM630, an effect that further underlines the agonist effect of CBC is CB2
receptor-mediated. AM630 is an inverse agonist at CB2 receptors (Ross et al, 1999), and
has previously been reported to increase (Grimsey et al., 2011), or have no effect (Atwood
et al., 2012), on CB2 surface receptor levels. Under our experimental conditions, AM630
did not have any appreciable effect on the cell surface receptors.
In the canonical view, GPCR signal desensitisation is usually mediated by GPCR kinase
(GRK)-mediated phosphorylation of GPCRs with phosphorylated receptors interacting
with arrestins to prevent further downstream signalling (Gainetdinov et al., 2004).
Information about the mechanisms of desensitisation of CB2 receptor signalling is sparse,
and the GRK involved in CBC-induced CB2 surface internalisation and desensitisation
have not been identified. Our results suggest that GRK2/3 kinases are likely not involved
in these processes, consistent with previous findings suggesting that GRK2/3 were not
likely involved in CB2 internalisation (Bouaboula et al., 1999).
Beta-caryophyllene, which is a terpenoid found in relative abundance within cannabis
and food plants, is a naturally occurring CB2-selective agonist (Gertsch et al., 2008). It
This article is protected by copyright. All rights reserved.

has both in vitro and in vivo CB2-mediated anti-inflammatory activities. Here, we have
shown that CBC, a phytocannabinoid, is also CB2-selective agonist. This selectivity
implies that CBC and/or its derivatives may be further investigated as a potential
therapeutic agent that influences the non- psychotropic CB2 receptor pathways of the
ECS. Understanding its mechanism of anti-inflammatory activity in-vitro and in-vivo, as
well as activity at other targets, would be valuable in developing its therapeutic potential.
Notably, the combination of CBC with THC produces enhanced anti- nociception and
anti-inflammatory responses in vivo (Davis & Hatoum, 1983; DeLong et al., 2010). This
may reflect pharmacokinetic interactions with THC, but also the pharmacodynamic
effects of CBC itself on inflammatory processes. Future research might further investigate
CBC in combination with THC and cannabidiol to formulate an optimal analgesic
cannabis-based medicine, with minor psychotropic effects and potentiated analgesia.
Acknowledgements
M.U is a recipient of the Macquarie University Postgraduate Research Scholarships for
International Students. We thank Dr Samuel Banister for his helpful comments on this
study.
Author contributions
M.U, M.S and M.C designed and analysed experiments. S.D synthesized CBC, M.U conducted
all other experiments. M.U, S.D, M.S, I.M and M.C prepared the manuscript. All authors have
seen the final paper.
This article is protected by copyright. All rights reserved.

Conflict of interest
The authors declare they have no conflicts of interest associated with this work.
Declaration of Transparency and Scientific Rigour
This Declaration acknowledges that this paper adheres to the principles for transparent
reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design
& Analysis, and Immunoblotting and Immunochemistry, and as recommended by funding
agencies, publishers and other organisations engaged with supporting research.
This article is protected by copyright. All rights reserved.

References
Alexander, S. P., Christopoulos, A., Davenport, A. P., Kelly, E., Marrion, N. V, Peters, J. A.,
… & Davies J.A.; CGTP Collaborators. (2017). THE CONCISE GUIDE TO
PHARMACOLOGY 2017/18: G
protein-coupled receptors. British Journal of Pharmacology, 174, S17–S129.
https://doi.org/10.1111/bph.13878
Atwood, B. K., Wager-Miller, J., Haskins, C., Straiker, A., & Mackie, K. (2012). Functional
Selectivity in CB2 Cannabinoid Receptor Signaling and Regulation: Implications for the
Therapeutic Potential of CB2 Ligands. Molecular Pharmacology, 81(2), 250–263.
https://doi.org/10.1124/mol.111.074013
Banister, S. D., Longworth, M., Kevin, R., Sachdev, S., Santiago, M., Stuart, J., …& Kassiou,
M. (2016). Pharmacology of Valinate and tert-Leucinate Synthetic Cannabinoids 5F-
AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA, MDMB-FUBINACA, MDMB-CHMICA,
and
Their
Analogues.
ACS
Chemical
Neuroscience,
7(9),
1241–1254.
https://doi.org/10.1021/acschemneuro.6b00137
Barann, M., Molderings, G., Brüss, M., Bönisch, H., Urban, B. W., & Göthert, M. (2002).
Direct inhibition by cannabinoids of human 5-HT3A receptors: probable involvement of an
allosteric modulatory site. British Journal of Pharmacology, 137(5), 589–596.
https://doi.org/10.1038/sj.bjp.0704829
Bayewitch, M., Rhee, M. H., Avidor-Reiss, T., Breuer, A., Mechoulam, R., & Vogel, Z. (1996).
(-)-Delta9-tetrahydrocannabinol antagonizes the peripheral cannabinoid receptor-mediated
This article is protected by copyright. All rights reserved.

inhibition of adenylyl cyclase. The Journal of Biological Chemistry, 271(17), 9902–9905.
https://doi.org/10.1074/jbc.271.17.9902
Bisogno, T., Ortar, G., Petrosino, S., Morera, E., Palazzo, E., Nalli, M., … & Di Marzo
V.;Endocannabinoid Research Group. (2009). Development of a potent inhibitor of 2-
arachidonoylglycerol hydrolysis with antinociceptive activity in vivo. Biochimica et
Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1791(1), 53–60.
https://doi.org/10.1016/j.bbalip.2008.10.007
Bolognini, D., Costa, B., Maione, S., Comelli, F., Marini, P., Di Marzo, V., … & Pertwee, R.
G. (2010). The plant cannabinoid Delta9-tetrahydrocannabivarin can decrease signs of
inflammation and inflammatory pain in mice. British Journal of Pharmacology, 160(3), 677–
687. https://doi.org/10.1111/j.1476-5381.2010.00756.x
Bonhaus, D. W., Chang, L. K., Kwan, J., & Martin, G. R. (1998). Dual Activation and
Inhibition of Adenylyl Cyclase by Cannabinoid Receptor Agonists: Evidence for Agonist-
Specific Trafficking of Intracellular Responses. The Journal of Pharmacology and
Experimental Therapeutics, 287, 884–888.
Bouaboula, M., Dussossoy, D., & Casellas, P. (1999). Regulation of peripheral cannabinoid
receptor CB2 phosphorylation by the inverse agonist SR 144528. Implications for receptor
biological responses. The Journal of Biological Chemistry, 274(29), 20397–20405.
https://doi.org/10.1074/JBC.274.29.20397
Cawston, E. E., Redmond, W. J., Breen, C. M., Grimsey, N. L., Connor, M., & Glass, M.
This article is protected by copyright. All rights reserved.

(2013). Real-time characterization of cannabinoid receptor 1 (CB1) allosteric modulators
reveals novel mechanism of action. British Journal of Pharmacology, 170(4), 893–907.
https://doi.org/10.1111/bph.12329
Curtis, M. J., Bond, R. A., Spina, D., Ahluwalia, A., Alexander, S. P. A., Giembycz, M. A., …
& McGrath, J. C. (2015). Experimental design and analysis and their reporting: new guidance
for publication in BJP. British Journal of Pharmacology, 172(14), 3461–3471.
https://doi.org/10.1111/bph.12856
Davis, W. M., & Hatoum, N. S. (1983). Neurobehavioral actions of cannabichromene and
interactions with Δ9-tetrahydrocannabinol. General Pharmacology, 14(2), 247–252.
https://doi.org/10.1016/0306-3623(83)90004-6
De Petrocellis, L., Ligresti, A., Moriello, A. S., Allarà, M., Bisogno, T., Petrosino, S., …& Di
Marzo, V. (2011). Effects of cannabinoids and cannabinoid-enriched Cannabis extracts on
TRP channels and endocannabinoid metabolic enzymes. British Journal of Pharmacology,
163(7), 1479–1494. https://doi.org/10.1111/j.1476-5381.2010.01166.x
De Petrocellis, L., Vellani, V., Schiano-Moriello, A., Marini, P., Magherini, P. C., Orlando, P.,
& Di Marzo, V. (2008). Plant-Derived Cannabinoids Modulate the Activity of Transient
Receptor Potential Channels of Ankyrin Type-1 and Melastatin Type-8. Journal of
Pharmacology
and
Experimental
Therapeutics,
325(3),
1007–1015.
https://doi.org/10.1124/jpet.107.134809
DeLong, G. T., Wolf, C. E., Poklis, A., & Lichtman, A. H. (2010). Pharmacological evaluation
This article is protected by copyright. All rights reserved.

of the natural constituent of Cannabis sativa, cannabichromene and its modulation by Δ9-
tetrahydrocannabinol. Drug and Alcohol Dependence, 112(1–2), 126–133.
https://doi.org/10.1016/J.DRUGALCDEP.2010.05.019
Deng, L., Guindon, J., Cornett, B. L., Makriyannis, A., Mackie, K., & Hohmann, A. G. (2015).
Chronic cannabinoid receptor 2 activation reverses paclitaxel neuropathy without tolerance or
cannabinoid receptor 1-dependent withdrawal. Biological Psychiatry, 77(5), 475–487.
https://doi.org/10.1016/j.biopsych.2014.04.009
Elsohly M and Gul W (2014) Constituents of cannabis sativa, in: Handbook of Cannabis
(Pertwee RG ed) pp. 3–22, Oxford University Press, Oxford, UK. ISBN 978‐0‐19‐966268‐5
https://doi.org/10.1093/acprof:oso/9780199662685.003.0001
Flores-Sanchez, I. J., & Verpoorte, R. (2008). Secondary metabolism in cannabis.
Phytochemistry Reviews, 7(3), 615–639. https://doi.org/10.1007/s11101-008-9094-4
Gainetdinov, R. R., Premont, R. T., Bohn, L. M., Lefkowitz, R. J., & Caron, M. G. (2004).
Desensitization of G protein–coupled receptors and neuronal functions. Annual Review of
Neuroscience, 27, 107–144. https://doi.org/10.1146/annurev.neuro.27.070203.144206
Gertsch, J., Leonti, M., Raduner, S., Racz, I., Chen, J.-Z., Xie, X.-Q., … & Zimmer, A. (2008).
Beta-caryophyllene is a dietary cannabinoid. Proceedings of the National Academy of Sciences,
105(26), 9099–9104. https://doi.org/10.1073/pnas.0803601105
Glass, M., & Felder, C. C. (1997). Concurrent stimulation of cannabinoid CB1 and dopamine
This article is protected by copyright. All rights reserved.

D2 receptors augments cAMP accumulation in striatal neurons: evidence for a Gs linkage to
the CB1 receptor. The Journal of Neuroscience, 17(14), 5327–5333.
https://doi.org/10.1523/JNEUROSCI.17-14-05327.1997
Grimsey, N. L., Goodfellow, C. E., Dragunow, M., & Glass, M. (2011). Cannabinoid receptor
2 undergoes Rab5-mediated internalization and recycles via a Rab11-dependent pathway.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1813(8), 1554–1560.
https://doi.org/10.1016/J.BBAMCR.2011.05.010
Guindon, J., Desroches, J., & Beaulieu, P. (2007). The antinociceptive effects of intraplantar
injections of 2-arachidonoyl glycerol are mediated by cannabinoid CB receptors. British
2
Journal of Pharmacology, 150(6), 693–701. https://doi.org/10.1038/sj.bjp.0706990
Harding SD, Sharman JL, Faccenda E, Southan C, Pawson AJ, Ireland S et al. (2018). The
IUPHAR/BPS Guide to PHARMACOLOGY in 2018: updates and expansion to encompass
the new guide to IMMUNOPHARMACOLOGY. Nucleic Acids Research, 46, D1091-D1106.
https://doi.org/10.1093/nar/gkx1121
Howlett, A.C (1987). Cannabinoid inhibition of adenylate cyclase: Relative activity of
constituents and metabolites of Marihuana. Neuropharmacology, 26(5), 507-512.
https://doi.org/10.1016/0028-3908(87)90035-9
Iannotti, F. A., Di Marzo, V., & Petrosino, S. (2016). Endocannabinoids and endocannabinoid-
related mediators: Targets, metabolism and role in neurological disorders. Progress in Lipid
Research, 62, 107–128. https://doi.org/10.1016/J.PLIPRES.2016.02.002
This article is protected by copyright. All rights reserved.

Ignatowska-Jankowska, B., Jankowski, M. M., & Swiergiel, A. H. (2011). Cannabidiol
decreases body weight gain in rats: Involvement of CB2 receptors. Neuroscience Letters,
490(1), 82–84. https://doi.org/10.1016/J.NEULET.2010.12.031
Ilan, A. B., Gevins, A., Coleman, M., ElSohly, M. A., & de Wit, H. (2005). Neurophysiological
and subjective profile of marijuana with varying concentrations of cannabinoids. Behavioural
Pharmacology, 16(5–6), 487–496. https://doi.org/10.1097/00008877-200509000-00023
Izzo, A. A., Borrelli, F., Capasso, R., Di Marzo, V., & Mechoulam, R. (2009). Non-
psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb. Trends
in Pharmacological Sciences, 30(10), 515–527. https://doi.org/10.1016/J.TIPS.2009.07.006
Kinsey, S. G., Long, J. Z., Cravatt, B. F., & Lichtman, A. H. (2010). Fatty acid amide hydrolase
and monoacylglycerol lipase inhibitors produce anti-allodynic effects in mice through distinct
cannabinoid receptor mechanisms. The Journal of Pain: 11(12), 1420–1428.
https://doi.org/10.1016/j.jpain.2010.04.001
La Rana, G., Russo, R., Campolongo, P., Bortolato, M., Mangieri, R. A., Cuomo, V., … &
Calignano, A. (2006). Modulation of Neuropathic and Inflammatory Pain by the
Endocannabinoid Transport Inhibitor AM404 [N-(4-Hydroxyphenyl)-eicosa-5,8,11,14-
tetraenamide]. Journal of Pharmacology and Experimental Therapeutics, 317(3), 1365–1371.
https://doi.org/10.1124/jpet.105.100792
Laprairie, R. B., Bagher, A. M., Kelly, M. E. M., & Denovan-Wright, E. M. (2015).
This article is protected by copyright. All rights reserved.

Cannabidiol is a negative allosteric modulator of the cannabinoid CB1 receptor. British Journal
of Pharmacology, 172(20), 4790–4805. https://doi.org/10.1111/bph.13250
Lauckner, J. E., Hille, B., & Mackie, K. (2005). The cannabinoid agonist WIN55,212-2
increases intracellular calcium via CB 1 receptor coupling to Gq/11 G proteins. Proceedings
of the National Academy of Sciences of the United States of America, 102(52):19144-19149.
https://doi.org/10.1073/pnas.0509588102
Lauckner, J. E., Jensen, J. B., Chen, H.-Y., Lu, H.-C., Hille, B., & Mackie, K. (2008). GPR55
is a cannabinoid receptor that increases intracellular calcium and inhibits M current.
Proceedings of the National Academy of Sciences of the United States of America, 105(7),
2699–2704. https://doi.org/10.1073/pnas.0711278105
Lee, Y.-R. & Wang, X. (2005). Concise Synthesis of Biologically Interesting (±)-
Cannabichromene, (±)-Cannabichromenic Acid, and (±)-Daurichromenic Acid. Bulletin of the
Korean Chemical Society. 26. 1933-1936. https://doi.org/10.5012/bkcs.2005.26.12.1933.
Lichtman, A. H., Shelton, C. C., Advani, T., & Cravatt, B. F. (2004). Mice lacking fatty acid
amide hydrolase exhibit a cannabinoid receptor-mediated phenotypic hypoalgesia. Pain,
109(3), 319–327. https://doi.org/10.1016/j.pain.2004.01.022
Ligresti, A., Moriello AS., Starowicz K., Matias I., Pisanti S., De Petrocellis L., …& Di Marzo
V. (2006). Antitumor Activity of Plant Cannabinoids with Emphasis on the Effect of
Cannabidiol on Human Breast Carcinoma. Journal of Pharmacology and Experimental
Therapeutics, 318(3), 1375–1387. https://doi.org/10.1124/jpet.106.105247
This article is protected by copyright. All rights reserved.

Longworth, M., Banister, S. D., Boyd, R., Kevin, R. C., Connor, M., McGregor, I. S., &
Kassiou, M. (2017). Pharmacology of Cumyl-Carboxamide Synthetic Cannabinoid New
Psychoactive Substances (NPS) CUMYL-BICA, CUMYL-PICA, CUMYL-5F-PICA,
CUMYL-5F-PINACA, and Their Analogues. ACS Chemical Neuroscience, 8(10), 2159–2167.
https://doi.org/10.1021/acschemneuro.7b00267
Lowe, J. D., Sanderson, H. S., Cooke, A. E., Ostovar, M., Tsisanova, E., Withey, S. L., … &
Bailey, C. P. (2015). Role of G Protein-Coupled Receptor Kinases 2 and 3 in µ-Opioid
Receptor Desensitization and Internalization. Molecular Pharmacology, 88(2), 347–356.
https://doi.org/10.1124/mol.115.098293
Maione, S., Piscitelli, F., Gatta, L., Vita, D., De Petrocellis, L., Palazzo, E., …& Di Marzo, V.
(2011). Non-psychoactive cannabinoids modulate the descending pathway of antinociception
in anaesthetized rats through several mechanisms of action. British Journal of Pharmacology,
162(3), 584–596. https://doi.org/10.1111/j.1476-5381.2010.01063.x
Mallipeddi, S., Janero, D. R., Zvonok, N., & Makriyannis, A. (2017). Functional selectivity at
G-protein coupled receptors: Advancing cannabinoid receptors as drug targets. Biochemical
Pharmacology, 128, 1–11. https://doi.org/10.1016/j.bcp.2016.11.014
Manzanares, J., Julian, M., & Carrascosa, A. (2006). Role of the cannabinoid system in pain
control and therapeutic implications for the management of acute and chronic pain episodes.
Current Neuropharmacology, 4(3), 239–257. https://doi.org/10.2174/157015906778019527
Mao, J., Price, D. D. ., Lu, J., Keniston, L., & Mayer, D.J . (2000). Two distinctive
This article is protected by copyright. All rights reserved.

antinociceptive systems in rats with pathological pain. Neuroscience Letters, 280(1), 13–16.
https://doi.org/10.1016/S0304-3940(99)00998-2
Marsicano, G., & Lutz, B. (1999). Expression of the cannabinoid receptor CB1 in distinct
neuronal subpopulations in the adult mouse forebrain. European Journal of Neuroscience,
11(12), 4213–4225. https://doi.org/10.1046/j.1460-9568.1999.00847.x
Mehmedic, Z., Chandra, S., Slade, D., Denham, H., Foster, S., Patel, A. S., … & ElSohly, M.
A. (2010). Potency trends of Δ9-THC and other cannabinoids in confiscated cannabis
preparations from 1993 to 2008. Journal of Forensic Sciences, 55(5), 1209–1217.
https://doi.org/10.1111/j.1556-4029.2010.01441.x
O’Sullivan, S. E., Tarling, E. J., Bennett, A. J., Kendall, D. A., & Randall, M. D. (2005). Novel
time-dependent vascular actions of Delta9-tetrahydrocannabinol mediated by peroxisome
proliferator-activated receptor gamma. Biochemical and Biophysical Research
Communications, 337(3), 824–831. https://doi.org/10.1016/j.bbrc.2005.09.121
Pertwee, R. G. (1999). Pharmacology of cannabinoid receptor ligands. Current Medicinal
Chemistry, 6(8), 635–664.
Potter, D. J., Clark, P., & Brown, M. B. (2008). Potency of Δ 9 –THC and Other Cannabinoids
in Cannabis in England in 2005: Implications for Psychoactivity and Pharmacology. Journal
of Forensic Sciences, 53(1), 90–94. https://doi.org/10.1111/j.1556-4029.2007.00603.x
Redmond, W. J., Cawston, E. E., Grimsey, N. L., Stuart, J., Edington, A. R., Glass, M., &
This article is protected by copyright. All rights reserved.

Connor, M. (2016). Identification of N-arachidonoyl dopamine as a highly biased ligand at
cannabinoid CB1 receptors. British Journal of Pharmacology, 173(1), 115–127.
https://doi.org/10.1111/bph.13341
Rhee, M. H., Vogel, Z., Barg, J., Bayewitch, M., Levy, R., Hanus, L., … & Mechoulam, R.
(1997). Cannabinol derivatives: binding to cannabinoid receptors and inhibition of
adenylylcyclase.
Journal
of
Medicinal
Chemistry,
40(20),
3228–3233.
https://doi.org/10.1021/jm970126f
Romano, B., Borrelli, F., Fasolino, I., Capasso, R., Piscitelli, F., Cascio, M., … & Izzo, A.
(2013). The cannabinoid TRPA1 agonist cannabichromene inhibits nitric oxide production in
macrophages and ameliorates murine colitis. British Journal of Pharmacology, 169(1), 213–
229. https://doi.org/10.1111/bph.12120
Rosenthaler, S., Pöhn, B., Kolmanz, C., Huu, C. N., Krewenka, C., Huber, A., … & Moldzio,
R. (2014). Differences in receptor binding affinity of several phytocannabinoids do not explain
their effects on neural cell cultures. Neurotoxicology and Teratology, 46, 49–56.
https://doi.org/10.1016/j.ntt.2014.09.003
Ross, R. A., Brockie, H. C., Stevenson, L. A., Murphy, V. L., Templeton, F., Makriyannis, A.,
& Pertwee, R. G. (1999). Agonist-inverse agonist characterization at CB1 and CB2
cannabinoid receptors of L759633, L759656, and AM630. British Journal of Pharmacology,
126(3), 665– 672. https://doi.org/10.1038/sj.bjp.0702351
Ryberg, E., Larsson, N., Sjögren, S., Hjorth, S., Hermansson, N.-O., Leonova, J., … &
This article is protected by copyright. All rights reserved.

Greasley,
P. J. (2007). The orphan receptor GPR55 is a novel cannabinoid receptor. British Journal of
Pharmacology, 152(7), 1092–1101. https://doi.org/10.1038/sj.bjp.0707460
Shinjyo, N., & Di Marzo, V. (2013). The effect of cannabichromene on adult neural
stem/progenitor
cells.
Neurochemistry
International,
63(5),
432–437.
https://doi.org/10.1016/j.neuint.2013.08.002
Shoemaker, J. L., Joseph, B. K., Ruckle, M. B., Mayeux, P. R., & Prather, P. L. (2005). The
Endocannabinoid Noladin Ether Acts as a Full Agonist at Human CB2 Cannabinoid Receptors.
The Journal of Pharmacology and Experimental Therapeutics, 314(2), 868–875.
https://doi.org/10.1124/jpet.105.085282
Soethoudt, M., Grether, U., Fingerle, J., Grim, T. W., Fezza, F., de Petrocellis, L., …& van der
Stelt, M. (2017). Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-
target activity. Nature Communications, 8, 13958. https://doi.org/10.1038/ncomms13958
Swift, W., Wong, A., Li, K. M., Arnold, J. C., & McGregor, I. S. (2013). Analysis of cannabis
seizures in NSW, Australia: cannabis potency and cannabinoid profile. PloS One, 8(7), e70052.
https://doi.org/10.1371/journal.pone.0070052
Sylantyev, S., Jensen, T. P., Ross, R. A., & Rusakov, D. A. (2013). Cannabinoid- and
lysophosphatidylinositol-sensitive receptor GPR55 boosts neurotransmitter release at central
synapses. Proceedings of the National Academy of Sciences, 110(13), 5193–5198.
https://doi.org/10.1073/pnas.1211204110
This article is protected by copyright. All rights reserved.

Turner, C. E., Elsohly, M. A., & Boeren, E. G. (1980). Constituests of Cannabis sativa L.
XVII. A review of the natural constituents. Journal of Natural Products, 43(2), 169–234.
https://doi.org/10.1021/np50008a001
Turner, C. E., & Elsohly, M. A. (1981). Biological activity of cannabichromene, its homologs
and
isomers.
Journal
of
Clinical
Pharmacology,
21(S1),
283S–291S.
https://doi.org/10.1002/j.1552-4604.1981.tb02606.x
This article is protected by copyright. All rights reserved.

Figure 1: Chemical structures of some phytocannabinoids
This article is protected by copyright. All rights reserved.